This invention relates to methods of using polynucleotides and polypeptides of the Fab family, as well as their variants, herein referred to as xe2x80x9cFabK,xe2x80x9d xe2x80x9cFabK polynucleotide(s),xe2x80x9d and xe2x80x9cFabK polypeptide(s),xe2x80x9d as the case may be.
Infections caused by or related to bacteria are a major cause of human illness worldwide, and the frequency of resistance to standard antibiotics has risen dramatically over the last decade. Hence, there exists an unmet medical need and demand for new anti-microbial agents against pathogenic bacteria, as well as drug screening methods to identify such agents.
An example of a bacterial enzyme that is resistant to a widely used antibacterial agent, Triclosan, is FabK. This enzyme, involved in fatty acid biosynthesis, has been recently reported from Streptococcus pneumoniae, a well-known human pathogen (Heath, et al. Nature 406: 145 (2000)). The specific activity of the enzyme under the published conditions was 64+/xe2x88x924 nmol minxe2x88x921, too low to efficiently screen for compounds that modulate the activity of the enzyme, such as inhibitors (Heath, et al. Nature 406: 145 (2000)). The present invention solves this problem by providing a method for screening for FabK agonists and antagonists, wherein FabK activity is sufficient to perform efficient compound screening.
A further problem identified by recent studies has been solved by this invention. Heath teaches that organisms expressing FabK will be refractory to FabI inhibitors, and that bacteria possessing both targets will require a combination of inhibitors to block growth (Heath, et al. Nature 406: 145 (2000)). The present invention provides methods of screening for compounds that inhibit both enzymes, as well as the use of such compounds as antimicrobial compounds.
The present invention relates methods using FabK, in particular FabK polypeptides and FabK polynucleotides, to screen for antimicrobial compounds.
Also provided by the invention is a method of screening for an agonist or antagonist of FabK polypeptide comprising the steps of: providing a reaction mixture comprising a FabK polypeptide; contacting a candidate compound to said reaction mixture; and detecting activation or inhibition of an activity of said FabK polypeptide.
Another aspect of the invention is a method for screening to identify compounds that activate or that inhibit an activity of Fab K polypeptide comprising a method selected from the group consisting of: (a) measuring the binding of a candidate compound to the polypeptide (or to the cells or membranes bearing the polypeptide) or a fusion protein thereof by means of a label directly or indirectly associated with the candidate compound; (b) measuring the binding of a candidate compound to the polypeptide (or to the cells or membranes bearing the polypeptide) or a fusion protein thereof in the presence of a labeled competitor; (c) testing whether the candidate compound results in a signal generated by activation or inhibition of the polypeptide, using detection systems appropriate to the cells or cell membranes bearing the polypeptide; or (d) mixing a candidate conmpound with a solution comprising a FabK polypeptide, to form a mixture, measuring activity of the polypeptide in the mixture, and comparing the activity of the mixture to a standard.
A still further aspect of the invention provides a method wherein said reaction mixture comprises crotonyl ACP and/or an ACP comprising a linked 6 to 8 carbon chain, and/or NADH and/or a cation.
Another aspect of the invention is a method wherein said detecting step comprises measuring a change in light absorption at least 2 time points.
The invention also provides a method wherein FabK polypeptide is an isolated polypeptide selected from the group consisting of: (i) an isolated polypeptide comprising an amino acid having at least 95% identity to the amino acid sequence of SEQ ID NO:2 over the entire length of SEQ ID NO:2; (ii) an isolated polypeptide comprising the amino acid sequence of SEQ ID NO:2, (iii) an isolated polypeptide that is the amino acid sequence of SEQ ID NO:2, and (iv) a polypeptide that is encoded by a recombinant polynucleotide comprising the polynucleotide sequence of SEQ ID NO:1.
Various changes and modifications within the spirit and scope of the disclosed invention will become readily apparent to those skilled in the art from reading the following descriptions and from reading the other parts of the present disclosure.